CN-116879209-B - Method for detecting CpG content in double-adjuvant recombinant novel coronavirus vaccine (pichia pastoris)

CN116879209BCN 116879209 BCN116879209 BCN 116879209BCN-116879209-B

Abstract

The invention relates to the field of CpG content detection methods, in particular to a method for detecting CpG content in a double-adjuvant recombinant novel coronavirus vaccine (pichia pastoris), which comprises the following steps of S1, taking 1ml of a novel coronavirus vaccine (pichia pastoris) test sample, loading into a10 mlEP tube, adding 1ml of 3% sodium hydroxide solution, ensuring that the sealing is perfect, heating to be clear through boiling water bath for 15 minutes, shaking uniformly, precisely measuring 100 μl of the mixed solution, loading into a10 mlEP tube, adding 4.9ml of water, shaking uniformly, taking the mixed solution as the test sample solution, and co-diluting by 100 times.

Inventors

ZHANG HAO
LIN MEIYU
GUO HAIFENG
GUO JING
ZHAO JIA
SHEN JIATIAN

Assignees

长春卓谊生物股份有限公司

Dates

Publication Date: 20260512
Application Date: 20230705

Claims (7)

1. The method for detecting the CpG content in the double-adjuvant recombinant novel coronavirus vaccine Pichia pastoris is characterized by comprising the following steps: S1, taking 1ml of a novel coronavirus vaccine Pichia pastoris test sample, putting the test sample into a 10ml EP tube, adding 1ml of 3% sodium hydroxide solution, ensuring that the test sample is sealed well, heating the test sample to be clear through boiling water bath for 15 minutes, shaking the test sample uniformly, precisely measuring 100 mu l of the mixed solution, putting the test sample into the 10ml EP tube, adding 4.9ml of water, shaking the test sample solution, diluting the test sample 100 times, S2, setting the wavelength of an ultraviolet-visible spectrophotometer to be 260nm, taking purified water as a blank control, measuring the absorbance values of a standard curve solution, a test sample solution and diluted antigen, S3, taking a standard curve of the absorbance value corresponding to the content mug/ml of the CpG standard solution, solving a linear regression equation Conc=aX+b, and taking the absorbance value of the measured sample into the linear regression equation, wherein the linear regression equation is as follows: 。
2. the method for detecting CpG content in Pichia pastoris as a double adjuvant recombinant novel coronavirus vaccine according to claim 1, wherein the CpG content detection further comprises a specificity test, and the adopted analysis method can accurately detect the capability of the detected object, and comprises the following specific steps: s1, precisely measuring 1ml of a test sample, loading the test sample into a 10ml EP tube, adding 1ml of 3% sodium hydroxide solution, ensuring that the test sample is well sealed, heating the test sample for 15 minutes by boiling water bath until the test sample is clear, shaking the test sample, precisely measuring 100 mu l of the mixed solution, loading the test sample into the 10ml EP tube, adding 4.9ml of water, shaking the test sample, diluting the test sample solution 100 times, S2, weighing 40 mu l of 2mg/ml CpG reference substance working solution, placing the working solution in a 5ml EP tube, adding purified water to supplement the working solution to 4ml, namely 20 mu g/ml CpG content, S3, preparing 1ml of antigen-aluminum hydroxide-sorbitol solution, preparing the antigen-aluminum hydroxide-sorbitol solution with the same content as the antigen, the aluminum hydroxide and the sorbitol in the test sample, weighing 50 μl of the mixed solution and 4.95ml of purified water, uniformly mixing, diluting 100 times, S4, determining the CpG content of the solution in the step S3 by adopting the steps S2-S3 in the claim 1.
3. The method for detecting CpG content in Pichia pastoris as a double adjuvant recombinant novel coronavirus vaccine according to claim 1, wherein the CpG content detection further comprises a linear test for determining the ability of the linear test result to directly proportional to the concentration of the detected substance in the sample within a designed range, and the method comprises the following specific steps: S1, measuring 20 mu l, 30 mu l, 40 mu l, 50 mu l and 60 mu l of CpG reference substance working solution of 2mg/ml, respectively placing into 5ml EP tube, adding purified water to make up to 4ml, namely, cpG content of 10 mu g/ml, 15 mu g/ml, 20 mu g/ml, 25 mu g/ml and 30 mu g/ml, S2, determining the CpG content of the solution in the step S1 by adopting the steps S2-S3 in the claim 1.
4. The method for detecting CpG content in Pichia pastoris as double adjuvant recombinant novel coronavirus vaccine according to claim 1, wherein the CpG content detection further comprises a test of repeatability and intermediate precision, the same homogeneous sample is measured under a specified measurement condition, the degree of proximity between the results obtained by the multiple sampling measurement is measured, the precision of the results obtained by the same analyst is referred to as repeatability, and the precision of the measurement results is changed under the same laboratory condition and is referred to as intermediate precision, and the method comprises the following specific steps: S1, taking a batch of test samples, putting 1ml of the test samples into a 10ml EP tube, adding 1ml of 3% sodium hydroxide solution, ensuring that the test samples are sealed well, heating the test samples for 15 minutes by boiling water bath until the test samples are clear, shaking the test samples uniformly, precisely measuring 100 mu l of the mixed solution, putting the test samples into a 10mlEP tube, adding 4.9ml of water, shaking the test samples uniformly, diluting the test samples 100 times as a test sample solution, taking 6 parallel samples for each test sample, S2, determining the CpG content of the solution in the step S1 by adopting the steps S2-S3 in the claim 1, and determining the relative standard deviation RSD value of the CpG content of 6 test sample solutions, S3, determining the CpG content of the solution in the step S1 by adopting the steps S2-S3 in the claim 1, and determining the CpG content RSD value of each test sample solution by an experimenter A and an experimenter B respectively.
5. The method for detecting CpG content in Pichia pastoris as a double adjuvant recombinant novel coronavirus vaccine according to claim 1, wherein the CpG content detection further comprises an accuracy test for determining the degree of approach of the result of the determination by the established method to a true value or a reference value, and the method is expressed as% recovery rate, and comprises the following specific steps: S1, preparing test seedlings with CpG concentration of 1mg/ml, 2mg/ml and 3mg/ml, taking 1ml of test sample, loading the test sample into a 10ml EP tube, adding 1ml of 3% sodium hydroxide solution, ensuring that the test sample is sealed well, heating the test sample for 15 minutes by boiling water bath until the test sample is clear, shaking the test sample, precisely measuring 100 mu l of mixed solution, loading the test sample into the 10ml EP tube, adding 4.9ml of water, shaking the test sample, taking the test sample as test sample solution, diluting the test sample 100 times, taking 3 parallel samples for each test sample, S2, determining the CpG content of the solution in the step S1 by adopting the steps S2-S3 in the claim 1.
6. The method for detecting the CpG content in the recombinant novel double-adjuvant coronavirus vaccine Pichia pastoris according to claim 1, wherein the CpG content detection further comprises a durability test, the tolerance degree of the test result is not affected when the test condition is slightly changed, the basis is provided for the established method to be used for routine test, the boiling time and the sodium hydroxide solution are changed, and the specific steps are as follows: s1, the difference between the step and the step S1 in the claim 1 is that the boiling time is changed to 14min and 16min, the concentration of the sodium hydroxide solution is changed to 2.8 percent and 3.2 percent, S2, determining the CpG content of the solution in the step S1 by adopting the steps S2-S3 in the claim 1.
7. The method for detecting CpG content in Pichia pastoris as a double adjuvant recombinant novel coronavirus vaccine according to any one of claims 1 to 6, wherein the vaccine comprises 1.5 mg/dose of aluminum hydroxide, 1.0 mg/dose of CpG, 10%/dose of sorbitol, 50. Mu.g/dose of antigen.

Description

Method for detecting CpG content in double-adjuvant recombinant novel coronavirus vaccine (pichia pastoris) Technical Field The invention relates to the field of CpG content detection methods, in particular to a method for detecting CpG content in a double-adjuvant recombinant novel coronavirus vaccine (pichia pastoris). Background CpGISS1018 is a 22-mer unmethylated CpG-B class oligonucleotide, cpGISS1018 stimulates B cell and NK cell activation. CpG-B localizes to transferrin receptor 1 (TFR 1) endosomes and leads to the production of IFN alpha through a TLR9 dependent mechanism, immunization with soluble CpG-B plus antigen induces activation of NF- κB to produce pro-inflammatory and Th1 cytokines by a mechanism dependent on TLR9 mediated activation of MyD88 in DCs. CpG is now a novel adjuvant that has been clinically proven to be safe and effective. CpGISS1018 is phosphorothioate backbone (PS) modification, where the sulfur atom replaces one of the non-bridging oxygen atoms in the phosphate backbone, and the PS modification is more hydrophobic and stable, and resists nuclease degradation after modification. The aluminum hydroxide adjuvant exists in the form of aluminum hydroxyl, is a fibrous particle, has an isoelectric point of about 10.0-11.0, exists in a solution with neutral pH as positively charged microparticles, can well absorb CpG with negative charges and negatively charged antigens, has lone pair electrons in amino acid, and can possibly generate coordination reaction by adding metal ions into the solution. Therefore, when the CpG content is measured, the adsorption reaction between aluminum hydroxide and CpG occurs, and when the CpG content is directly measured, the amount to be measured becomes smaller than the amount to be charged, and the measurement value becomes lower. Disclosure of Invention The invention aims to provide a method for detecting CpG content in double-adjuvant recombinant novel coronavirus vaccine (pichia pastoris), which solves the problems. In order to achieve the above purpose, the following technical scheme is provided: the method for detecting the CpG content in the double-adjuvant recombinant novel coronavirus vaccine (pichia pastoris) comprises the following steps: S1, 1ml of a novel coronavirus vaccine (pichia pastoris) test sample is taken and filled into a 10mlEP tube, 1ml of 3% sodium hydroxide solution is added, after sealing is ensured to be complete, the test sample is heated to be clear through boiling water bath for 15 minutes, shaking is carried out, 100 μl of the mixed solution is precisely measured and filled into a 10mlEP tube, 4.9ml of water is added, shaking is carried out, and the test sample solution is taken and diluted 100 times altogether. S2, setting the wavelength of an ultraviolet-visible spectrophotometer to be 260nm, and measuring the absorbance values of a standard curve solution, a sample solution and diluted antigen by taking purified water as a blank control. S3, taking the absorbance value corresponding to the CpG standard solution content (mug/ml) as a standard curve, solving a linear regression equation (Conc=aX+b), and taking the absorbance value of the measured sample into the linear regression equation. Preferably, the CpG content detection further includes a specificity test, so as to ensure that the adopted analysis method can accurately detect the ability of the detected object under the condition that other components (such as impurities, degradation products, auxiliary materials, etc.) may exist, and specifically comprises the following steps: S1, precisely measuring 1ml of a test sample, loading the test sample into a 10mlEP pipe, adding 1ml of 3% sodium hydroxide solution, ensuring that the test sample is well sealed, heating the test sample for 15 minutes by using boiling water bath until the test sample is clear, shaking the test sample, precisely measuring 100 mu l of the mixed solution, loading the test sample into a 10mlEP pipe, adding 4.9ml of water, shaking the test sample, and diluting the test sample 100 times. S2, measuring 40 mu l of 2mg/mlCpG control working solution, placing the working solution in a 5mlEP tube, adding purified water to supplement the working solution to 4ml, namely 20 mu g/ml CpG. S3, preparing 1ml of antigen-aluminum hydroxide-sorbitol solution (blank solution), preparing the antigen-aluminum hydroxide-sorbitol solution to be consistent with the contents of the antigen, the aluminum hydroxide and the sorbitol in the test sample, weighing 50 μl of the mixed solution and 4.95ml of purified water, uniformly mixing, and diluting 100 times. S4, determining the CpG content of the solution in the step S3 by adopting the steps S2-S3 in the claim 1. Preferably, the CpG content detection further comprises a linear test for determining the ability of the linear test result to directly scale with the concentration of the analyte in the sample within a designed range, and the specific steps are as follows: